Coding

Part:BBa_K2872019

Designed by: Dina Altarawneh   Group: iGEM18_CMUQ   (2018-10-10)
Revision as of 20:04, 20 October 2021 by Ellie Malkopoulou (Talk | contribs)


Cas12a protein


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1198
    Illegal PstI site found at 1696
    Illegal PstI site found at 3403
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1198
    Illegal PstI site found at 1696
    Illegal PstI site found at 3403
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 938
    Illegal BglII site found at 1271
    Illegal BglII site found at 1841
    Illegal BglII site found at 2108
    Illegal BglII site found at 2729
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1198
    Illegal PstI site found at 1696
    Illegal PstI site found at 3403
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1198
    Illegal PstI site found at 1696
    Illegal PstI site found at 3403
    Illegal NgoMIV site found at 1000
    Illegal NgoMIV site found at 1999
    Illegal NgoMIV site found at 2701
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1329
    Illegal SapI.rc site found at 1389

Part Information

This part was synthesized using Cas12a DNA coding sequence from pY016 (pcDNA3.1-hLbCpf1) plasmid (Addgene plasmid # 69988) [1]

Biology and Usage

Cas12a is a programmable DNA endonuclease guided by a single guide RNA (gRNA) and can be used in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules [2]

  • Targeting requires a gRNA complementary to the target site as well as a 5' TTTN protospacer adjacent motif (PAM) on the DNA strand opposite the target sequence.
  • Cleavage occurs ~18 bases 3' of the PAM and leaves 5' overhanging ends.
  • Cas12 a relies on a short (40-44 base) guide RNA to initiate DNA degradation.
  • Cas12a is active from 16 to 48°C and maintains activity at lower temperatures than the Acidaminococcus orthologs, permitting editing in ectothermic organisms such as zebra fish and xenopus.
  • High concentration liquid format can be used for microinjection, electroporation and lipofection [3]

There are 2 classes of effectors in the microbial adaptive immune system; class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Cas12a (Cpf1) is a putative class 2 CRISPR effector which mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. We chose Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) -along with Acidaminococcus- because it was found to have efficient genome-editing activity in human cells. [4]


Source

Lachnospiraceae bacterium ND2006

References

[1] Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 10.1016/j.cell.2015.09.038. 10.1016/j.cell.2015.09.038 PubMed 26422227
[2] Chen, J. S., Ma, E., Harrington, L. B., Costa, M. D., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018, April 27). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Retrieved from http://science.sciencemag.org/content/360/6387/436
[3] Biolabs, N. E. (n.d.). EnGen® Lba Cas12a (Cpf1). Retrieved from https://international.neb.com/products/m0653-engen-lba-cas12a-cpf1#Product Information_Notes
[4] Lassner, M. (2017). Faculty of 1000 evaluation for Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. F1000 - Post-publication Peer Review of the Biomedical Literature. doi:10.3410/f.725812264.793537694


Effect of ions on Cas12a’s collateral cleavage activity

iGEM Thrace 2021

Cas12as’ cleavage activity is highly dependent on the presence or absence of specific ions. Not all orthologs of Cas12a react in the same way to the presence of these ions. In fact, there are some major differences between them.

  • FnoCas12a
    • Has the ability, in the presence of Mg2+, to cleave DNA without the need of a guide RMA.
    • Cleaves ssDNA more efficiently at lower concentrations of Mn2+ compared to dsDNA.
    • In FnoCas12a the ds plasmid nicking activity is promoted also by Co2+
  • AsCas12a
    • Is able to degrade DNA substrate in the presence of Mg2+ and/or Mn2+.
    • Mg2+ is required for a crRNA free cleavage activity of DNA substrate. Furthermore, in the presence of Mg2+ buffer, AsCas12a, displays a preferred endonuclease activity for ssDNA rather than dsDNA.
    • For circular ssDNA, AsCas12a enzyme has the same activity for the same period of time, no matter which kind of metal ion (Mg2+ or Mn2+) was used
    • Co2+ also promotes the cleavage of ssDNA in the case of AsCas12a
  • LbCas12a
    • The crRNA-independent DNase activity of LbCas12a can be activated only by Mn2+
    • Mn2+ is not able to trigger LbCas12a-mediated dsDNA cleavage activity.
    • The nickase activity of LbCas12a after being incubated in Mn2+ for 1 min is comparable with that of AsCas12a with 30-min incubation in Mg2+
    • None of the divalent metal ions is able to trigger LbCas12a-mediated dsDNase activity
  • FnCas12a
    • Mn2+ is able to trigger FnCas12a-mediated dsDNA cleavage activity.
    • CrRNA-independent DNase activity of FnCas12a , for both dsDNA and ssDNA,can be activated only in the presence of Mn2+.

References

1. Sundaresan, R., Parameshwaran, H. P., Yogesha, S. D., Keilbarth, M. W., & Rajan, R. (2017). RNA-independent DNA cleavage activities of Cas9 and CAS12A. Cell Reports, 21(13), 3728–3739. https://doi.org/10.1016/j.celrep.2017.11.100 ‘
2. Dai, Y., Somoza, R. A., Wang, L., Welter, J. F., Li, Y., Caplan, A. I., & Liu, C. C. (2019). Exploring the trans‐cleavage activity of CRISPR‐CAS12A (cpf1) for the development of a universal electrochemical biosensor. Angewandte Chemie, 131(48), 17560–17566. https://doi.org/10.1002/ange.201910772
3. Li, B., Yan, J., Zhang, Y., Li, W., Zeng, C., Zhao, W., Hou, X., Zhang, C., & Dong, Y. (2020). CRISPR-CAS12A possesses unconventional DNase activity that can be inactivated by synthetic oligonucleotides. Molecular Therapy - Nucleic Acids, 19, 1043–1052. https://doi.org/10.1016/j.omtn.2019.12.038

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